Abstract B076: βIII-Tubulin is a brake on extrinsic cell-death in pancreatic cancer
Authors: John Kokkinos, George Sharbeen, Janet Youkhana, Cyrille Boyer, David Goldstein, Koroush S. Haghighi, Joshua A. McCarroll, Phoebe A. Phillips
Journal: Cancer Research (2022)

Abstract Background: Pancreatic Ductal Adenocarcinoma (PDAC) urgently needs more effective therapies, with a 5-year survival rate &amp;lt;11%. We previously demonstrated βIII-tubulin knockdown in PDAC cells reduced tumor growth and metastasis in vivo, increased chemosensitivity, and induced apoptosis, making it a potent therapeutic target [McCarroll et al, Oncotarget, 6, 2235 (2015)]. However, mechanisms driving these effects are unknown. Apoptosis consists of intrinsic (caspase-9) and extrinsic (caspase-8) pathways. To better understand mechanism and inform therapeutic combinations, our study investigated apoptotic pathways controlled by βIII-tubulin. Aims: 1) Evaluate apoptotic pathways controlled by βIII-tubulin in PDAC cells a) in vitro and b) in vivo; 2) Assess the effect of silencing βIII-tubulin combined with tumor-specific extrinsic apoptosis inducer (TRAIL) in a patient-derived 3D PDAC model. Methods: 1a) PDAC cells were transfected with control-siRNA or βIII-tubulin-siRNA ± caspase-8 or -9 inhibitors and apoptosis measured. The effect of βIII-tubulin knockdown on PDAC cell sensitivity to extrinsic apoptosis inducers (TRAIL, TNFα, FasL) was tested (n≥3). TRAIL induces extrinsic cell death by binding to the TRAIL-receptor DR5, inducing clustering and intracellular activation of caspase 8. DR5 localization following βIII-tubulin knockdown was assessed by confocal microscopy (n=4). Live cell imaging was used to assess trafficking of GFP-tagged DR5 at the membrane of PDAC cells (n≥17 cells/treatment). 1b) Mice with orthotopic PDAC tumors (n=10/group) were treated (5-weeks post-implantation) with nanoparticle+βIII-tubulin-siRNA or control-siRNA for 3.5-weeks. Tumor volume and cleaved caspase-8 were measured. 2) Explants [validated patient-derived 3D tumor model with complete intact stroma, as described in: Kokkinos et al, Sci Rep, 11,1941 (2021); Sharbeen et al, Cancer Res, 81,3461 (2021)] from surgically resected PDAC tumors (5 patients) were cultured for 12 days and treated with nanoparticle+βIII-tubulin-siRNA or control-siRNA every 3 days and TRAIL once weekly. Tumor explants were fixed and assessed for tumor and viability markers via immunohistochemistry. Results: 1a) βIII-tubulin knockdown-induced apoptosis in PDAC cells was blocked by inhibiting caspase-8, but not caspase 9. Knockdown of βIII-tubulin sensitized PDAC cells to extrinsic apoptosis inducers. βIII-tubulin knockdown increased the movement of TRAIL-receptor DR5 through the cell membrane and triggered its clustering in PDAC cells. 1b) βIII-tubulin knockdown in orthotopic PDAC mouse tumors decreased tumor growth and activated extrinsic apoptosis. 2) In patient-derived tumor explants, βIII-tubulin-siRNA plus TRAIL reduced tumor cell frequency. Conclusions: 1) Silencing βIII-tubulin in PDAC cells sensitizes them to extrinsic apoptosis inducers and facilitates increased membrane clustering of the TRAIL receptor DR5. 2) βIII-tubulin knockdown represents an innovative therapeutic strategy to unleash a suicide signal in PDAC cells and render them sensitive to a tumor-specific therapeutic. Citation Format: John Kokkinos, George Sharbeen, Janet Youkhana, Cyrille Boyer, David Goldstein, Koroush S. Haghighi, Joshua A. McCarroll, Phoebe A. Phillips. βIII-Tubulin is a brake on extrinsic cell-death in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B076.